Ecological and Evolutionary Physiology of the Stress Response and Stress Proteins

总体目标：拟议的活动继续一个研究计划，其目标是了解复杂的生理和生化机制的起源和维持，使生物体能够在具有挑战性的环境中生存。该程序使用热休克蛋白Hsp70作为模型系统，它可以最大限度地减少应激引起的其他蛋白质损伤，并在果蝇中编码它的基因。未解决的主要问题是：自然种群中Hsp70水平变异的遗传基础是什么？这种变异是如何产生的？先前的研究表明，转座因子，即可以从一个基因转移到另一个基因的DNA序列，是这些问题的答案。这项研究的主要目的是验证这一假设。具体目标：首先，该研究将确定转座因子实际插入编码热休克蛋白的基因的频率，并将该频率与其他在结构、组织和调控上与热休克基因相似或不相似的基因的频率进行比较。这样做，研究将发现许多转座因子自然插入热休克基因的实例。接下来，研究人员将利用这些天然突变体来检查这些插入是否真的对热休克基因有预期的影响：信使RNA、热休克蛋白的减少，以及抵抗压力的能力。最后，研究人员将进行“实验室进化”，在这种进化中，果蝇菌株在有利于保留或消除热休克基因中有或没有转座因子的个体的环境中竞争。最后的实验将测试热休克基因中的转座因子是否真的对个体适应性有预期的影响。方法：(a)生物信息学，分析已有的信息来预测基因的特性。(b)高通量筛选，同时快速分析许多种群中的许多基因，以发现遗传事件。这将使用一种克隆基因的新技术“通用快走”。(c)转录谱分析，对特定基因表达时产生的信使rna进行定量分析。这种分析将使用两种技术，一种是将热休克基因的调控区域与基因表达时发光的报告蛋白融合，另一种是定量实时聚合酶链反应（quantitative real-time PCR），通过定量信使RNA在化学反应中的积累来测量信使RNA。(d)如上所述的实验室进化。意义：该研究的独特之处在于，它将阐明一种明显反复导致蛋白质表达减少并具有适应性后果的机制。由于对所讨论的基因/蛋白质进行了广泛的背景研究，它可以严格地确定调控突变和它们所调节的蛋白质水平的变化的重要性。这项拟议的研究有可能成为一项具有里程碑意义的研究，在这项研究中，所有在变异的进化产生、基因序列、细胞功能、有机体功能、适应性、种群中基因频率和对自然选择的反应之间经常缺失的联系都得到了解释。

Broad goals: The proposed activity continues a research program whose goal is to understand the origin and maintenance of complex physiological and biochemical mechanisms that enable organisms to persist in challenging environments. This program uses as a model system the heat-shock protein Hsp70, which minimizes stress-induced damage of other proteins, and the genes that encode it in the fruit fly, Drosophila. The principal remaining unanswered questions are: What is the genetic basis for the variation in Hsp70 levels among natural populations, and how does this variation arise? Prior work suggests that transposable elements, DNA sequences that can move from gene to gene, are the answer to these questions. The major goal of the research is to test this hypothesis.Specific aims: First the research will determine the frequency with which transposable elements actually insert in the genes encoding heat-shock proteins, and compare this frequency with that for other genes that either do or do not resemble heat-shock genes in their structure, organization, and regulation. In so doing, the research will discover numerous instances of transposable elements naturally inserting into heat-shock genes. Next, the research will use these natural mutants to examine whether the insertions actually have the expected effect on heat-shock genes: reduction in messenger RNA, heat-shock protein, and ability to withstand stress. Finally, the researchers will undertake "laboratory evolution", in which fruit fly strains compete in environments that ought to favor retention or elimination of individuals with and without transposable elements in their heat-shock genes. This final experiment would test whether transposable elements in the heat-shock genes actually have their expected effect on individual fitness.Approaches: (a) Bioinformatics, the analysis of pre-existing information to predict genes' properties. (b) High-throughput screening, the simultaneous and rapid analysis of many genes in many populations to detect genetic events. This will use "universal fast walking", a new technique for cloning genes. (c) Transcriptional profiling, the quantification of messenger RNAs produced by specific genes when they are expressed. This profiling would use two techniques, the fusion of the heat-shock genes' regulatory region with a reporter protein that emits light when the gene is expressed, and quantitative real-time PCR, which measures messenger RNA by quantifying its accumulation in a chemical reaction. (d) Laboratory evolution, as described above.Significance: The proposed research is distinctive in that it will elucidate a mechanism that apparently has repeatedly resulted in the decreased expression of a protein, with adaptive consequences. Because of extensive background work on the genes/protein in question, it can rigorously assign significance to both regulatory mutations and variation in the level of the protein they regulate. The proposed research has the potential to become a landmark study in which all of the often-missing links among evolutionary generation of variation, gene sequence, cellular function, organismal function, fitness, gene frequency in populations, and response to natural selection are explained.